Phosphatidic acid, which serves as the backbone of glycerophospholipid biosynthesis, is synthesized de novo by the sequential acylation of glycerol-3-phosphate. Glycerol-3-phosphate acyltransferase (GPAT) acylates the sn-1 position to form 1-acylglycerol-3-phosphate (lysophosphatidate) and this is the first committed and presumed to be rate-limiting, step in glycerophospholipid synthesis. 1-Acylglycerol-3-phosphate acyltransferase (AGPAT) acylates the sn-2 position to generate 1,2-diacylglycerol-3-phosphate (phosphatidate). Both GPAT and AGPA activities are found in the endoplasmic reticulum. In addition, GPAT that prefers saturated fatty acyl-CoA is found in mitochondria. Fatty acyl-CoA preferences of the mitochondrial GPAT and microsomal AGPAT may play key roles in the asymmetric distribution of fatty acids, ie, saturated at the sn-1 position and unsaturated at the sn-2 position, which is considered to be of major importance in maintaining the functional and structural function of glycerophospholipids. The applicant cloned the murine mitochondrial GPAT, expressed the protein in insect cells, and purified and reconstituted the GPAT activity. Recently, the applicant also cloned a full-length mammalian cDNA sequence homologous to known AGPATs from other species, and its mRNA is highly expressed in testis and brain, tissues where long chain polyenoic acids are abundant. The applicant is now in a unique position to address, by biochemical and molecular biological approaches, the role of these two acyltransferases in the positional distribution of fatty acids in glycerophospholipids and in the regulation of lipid metabolism. There are three specific aims. 1. The applican will determine the fatty acyl-CoA binding site of the mitochondrial GPAT by in vitro mutagenesis and photo-affinity labeling. The investigator will generate cells either depleted of, or expressing different levels of, the mitochondrial GPAT. 2. Using these cells, the applicant will determine the positional fatty acid composition of glycerophospholipids to demonstrate the role of mitochondrial GPAT in the prevalence of saturated fatty acids at the sn-1 position. The investigator will also measure fatty acid oxidation and esterification to examine the role of mitochondrial GPAT in lipid metabolism. The applicant will positively identify and characterize the cloned AGPAT by complementing an E. coli mutant and analyzing fatty acid composition. She will also express this AGPAT in acyl-CoA. She will also determine the contribution of this AGPAT in the enrichment of long chain polyenoic acids at the sn-2 position of glycerophospholipids.